There has long been an urgent need in the medical community for a non-toxic blood substitute suitable for transfusion into a patient. In addition to trauma victims and surgical patients, patients suffering from diseases such as hemophilia and sickle-cell anemia are in need of frequent transfusions. One in twenty Americans will need a blood transfusion at some point in their lives, and each year approximately eight million volunteer donors make approximately 14 million blood donations. Several shortcomings of donated blood have contributed to the urgent demand for a cell-free blood substitute, including: the need to match blood types; concerns regarding disease transmission; morally- or religiously-based objections to blood transfusion; requirement for freezing of blood units; and limited storage lifetime. There are currently no clinically utilized oxygen-carrying blood substitutes for humans.
Hemoglobin is the molecule found within red blood cells that chelates molecular oxygen in the lungs and transports it throughout the body. The idea of using hemoglobin-containing solutions as a cell-free blood substitute has always been appealing. However, clinical studies consistently revealed that cell-free hemoglobin was associated with vasoconstriction leading to bradycardia, hypertension and renal failure. Infusion of these products increases the risk of myocardial infarction and death in human clinical trials. Many of these toxicities have now been attributed to the reaction with and scavenging of endogenous nitric oxide (NO), an important blood vessel dilator elaborated by the lining cells (endothelium) of the blood vessels. A major focus of recent research has been to attempt to modify hemoglobin in such a way that mitigates its NO scavenging and toxicity. For example, hemoglobin has been polymerized, intra-molecularly cross-linked, and conjugated to polyethyleneglycol (PEG). This work has resulted in some improvements in cell-free hemoglobin that increase circulating half lives, modulate oxygen affinities and ameliorate renal toxicity. However, vascular toxicity associated with cell-free hemoglobin remains a concern and is thought to be largely mediated by scavenging of nitric oxide and development of hypertension, inflammation and platelet aggregation. Of great clinical benefit would be the ability to resuscitate trauma or surgical patients with a cell-free hemoglobin solution that improves oxygen delivery without causing hypertension.
Recent studies reveal that the ubiquitous circulating anion nitrite (NO2−) is a vasodilator and intrinsic signaling molecule (Gladwin et al., Proc. Natl. Acad. Sci. USA 97:11482-11487, 2000; Cosby et al., Nat. Med. 9:1498-1505, 2003; Gladwin et al., Nature Chemical Biology 1:308-314, 2005; Bryan et al., Nature Chemical Biology 1:290-297, 2005; Modin et al., Acta Physiologica Scandinavica 171:9-16, 2001). The vasodilator activity of nitrite is associated with an allosterically controlled heme-based reduction of nitrite to nitric oxide (NO) by deoxygenated hemoglobin (deoxyHb) (Huang et al., J. Biol. Chem. 280:31126-31131, 2005; Huang, et al., J. Clin. Invest. 115:2099-2107, 2005). Nitrite infusions into the human circulation increase blood flow at near-physiological concentrations (Cosby et al., Nat. Med. 9:1498-1505, 2003). This vasodilation is temporally associated with increases in red cell heme iron-nitrosylated hemoglobin (HbFeII—NO, designated as {FeNO}7 using the Enemark-Feltham notation; Enemark & Feltham, Coordination Chemistry Reviews 13:339-406, 1974) and to a lesser extent S-nitrosated hemoglobin (SNO-Hb, hemoglobin nitrosated at the β-93 cysteine; Cosby et al., Nat. Med. 9:1498-1505, 2003).